Fluorinated Arylamide Derivatives

Example 1

Synthesis

[0280]The compounds of the present invention were prepared by the general methods outlined in the synthetic schemes below, as exemplified below.

A. Benzothiophenes

[0281]A1. Compounds from (Carboxy-fluoro-methyl)-benzothiophenes.

[0282]Scheme 1 illustrates the use of (carboxy-fluoro-methyl)-benzothiophenes to generate amides, and various heterocycles.

A2. Compounds from (Carboxy-difluoro-methyl)-benzothiophenes.

[0283]Scheme 2 illustrates the use of (carboxy-difluoro-methyl)-benzothiophenes to generate amides, and various heterocycles.

B. Benzamides

[0284]B1. Compounds from (Carboxy-fluoro-methyl)-benzoic acids.

[0285]Scheme 3 illustrates the use of (carboxy-fluoro-methyl)-benzoic acids to generate amides, and various heterocycles.

B2. Compounds from (Carboxy-difluoro-methyl)-benzoic acids.

[0286]Scheme 4 illustrates the use of (carboxy-difluoro-methyl)-benzoic acids to generate amides, and various heterocycles.

B3. Compounds from (Fluoro-alkoxycarbonyl-alkyl)-benzoic acids.

[0287]Scheme 5 illustrates the use of (fluoro-alkoxycarbonyl-alkyl)-benzoic acids to generate amides, and various heterocycles.

Synthesis of Diaminoarylpyrazole

[0288]

Experimental

[0289]Procedures for A1. Compounds from (carboxy-fluoro-methyl)-benzothiophenes.

[0290]Ethyl 6bromo-1-benzothiophene-2-carboxylate. Sodium hydride (60% dispersion in mineral oil, 0.73 g, 18.3 mmol) was suspended in DMSO (10 mL) and ethyl mercaptoacetate (1.11 mL, 10.1 mmol) was added potionwise using a water bath to moderate the exotherm. On complete addition, the water bath was removed and stirring continued for 15 minutes. A solution of 4-bromo-2-fluorobenzaldehyde (1.86 g, 9.16 mmol) in DMSO (2 mL) was added in one portion. The dark solution was stirred for 15 minutes before pouring into cold water (300 mL). The products were extracted into Et2O (2×200 mL). The combined organic extracts were washed with brine, dried over MgSO4 and concentrated in vacuo. Purification of the residue by MPLC gave the desired product (pale yellow solid). 1H NMR (DMSO-d6) δ 8.37 (d, J=1.8 Hz, 1H), 8.17 (s, 1H), 7.94 (d, J=8.4 Hz, 1H), 7.60 (dd, J=8.4, 1.8 Hz, 1H), 4.32 (q, J=7.2 Hz, 2H), 1.30 (t, J=7.2 Hz, 3H).

[0291]Di-tert-butyl [2-(ethoxycarbonyl)-1-benzothien-6-yl]malonate. Di-tert-butyl malonate (1.5 g, 6.93 mmol) was dissolved in THF (6 mL) and sodium hydride (60% dispersion in mineral oil, 0.28 g, 7.00 mmol) was added. The mixture was stirred for 10 minutes before adding Pd(P2Bu3)2 (0.1 g, 0.196 mmol) and a solution of ethyl 6-bromo-1-benzothiophene-2-carboxylate (1.8 g, 6.31 mmol) in THF (12n-L). The resulting mixture was heated to reflux under N2 for 18 hours. Room temperature was attained, saturated NH4Cl (150 mL) was added and the products extracted into EtOAc (2×125 mL). The combined organic extracts were dried over MgSO4 and concentrated in vacuo. Purification of the residue by MPLC gave the desired product (pale yellow solid). 1H NMR (DMSO-d6) δ 8.17 (s, 1H), 8.01 (s, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.45 (dd, J=8.4, 1.8 Hz, 1H), 4.83 (s, 1H), 4.33 (q, J=7.2 Hz, 2H), 1.40 (s, 18H), 1.31 (t, J=7.2 Hz, 3H).

[0292]2-(2-Ethoxycarbonyl-benzo[b]thiophen-6-yl)-2-fluoro-malonic acid di-tert-butyl ester. To a suspension of sodium hydride (0.96 g, 24.0 mmol) in THF (75 mL) cooled to 0° C. was added a solution of di-tert-butyl [2-(ethoxycarbonyl)-1-benzothien-6-yl]malonate (described above) (9.66 g, 23.0 mmol) in THF (150 mL) under nitrogen. The resulting orange solution was stirred at 4° C. for 15 minutes. DMF (225 mL) was added followed by Selectfluor (8.15 g, 23-0 mmol). The reaction was warmed to ambient temperature and stirred for 4 hours under nitrogen. The reaction was quenched with ammonium chloride solution and partitioned between ethyl acetate and water. The organics were washed with water and brine, dried over magnesium sulfate, filtered and evaporated in vacuo. Purification by flash column chromatography (2-20% ethyl acetate/hexanes) gave a mixture of starting material and product. This was resubjected to above reaction conditions and purified in a similar manner to give a pale yellow solid. 1H NMR (DMSO-d6) δ 8.21 (s, 1H), 8.17 (s, 1H), 8.07 (d, J=8.8 Hz, 1H), 7.52 (d, J=9.1 Hz, 1H), 4.33 (q, J=7.3 Hz, 2H), 1.44 (s, 18H), 1.31 (t, J=7.0 Hz, 3H).

[0293]6-(Carboxy-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester. Di-tert-butyl [2-(ethoxycarbonyl)-1-benzothien-6-yl](fluoro)malonate (9.3 g) was stirred in DCM (40 mL)/TFA (20 mL) at room temperature overnight. The solvent was removed in vacuo and the residue partitioned between saturated NaHCO3-EtOAc. The aqueous phase was acidified with 2N HCl and extracted with EtOAc. The combined organic extracts were dried over MgSO4 and concentrated in vacuo. The residue was suspended in H2O (50mL) and heated to reflux for 2 hours. Room temperature was attained and the products extracted into EtOAc. The combined organic extracts were dried over MgSO4 and concentrated in vacuo to give the product as a pale yellow solid. 1H NMR (DMSO-d6) δ 13.60 (br s, 1H), 8.22 (s, 1H), 8.18 (s, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.51 (d, J=8.4 Hz, 1H), 6.14 (d, J=47.4 Hz, 1H), 4.35 (q, J=7.2 Hz, 2H), 1.33 (t, J=7.2 Hz, 3H).

[0294]6-(Carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester. To a slurry of 6-(carboxy-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester (400 mg, 1.42 mmol) in DCM (10 mL) was added oxalyl chloride (0.148 mL, 1.70 mmol) and 2 drops of DMF. After 20 min, the resultant solution was added to a solution of NH4OH (0.9 mL, 7.08 mmol) in DCM (5 mL) dropwise. After 1 h, the solvent was removed in vacuo and the solid was triturated with MeOH and H2O. The white solid was filtered and used without further purification. 1H NMR (DMSO-d6) δ 8.20 (s, 1H), 8.11 (s, 1H), 8.04 (d, J=8.2 Hz, 1H), 7.91 (br s, 1H), 7.62 (br d, J=2.4 Hz, 1H), 7.51 (d, J=8.2 Hz, 1H), 5.94 (d, J=45.7 Hz, 1H), 4.33 (q, J=7.2 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H). cal'd 282 (MH+), exp 282 (MH+).

[0295]6-(Carbamoyl-fluoro-methyl)benzo[b]thiophene-2-carboxylic acid. To a solution of 6-(carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester (295 mg, 1.05 mmol) in MeOH/HF (1.5/6 mL) was added 1M LiOH (1.16 mL). The resultant solution was stirred overnight and 1N HCl was added dropwise to acidify (˜2 mL). The solvent was removed in vacuo and the solid was used without further purification. cal'd 254 (MH+), exp 254 (MH+).

6-(Carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-2-yl-phenyl)amide

[0296]Step 1: Coupling; To a solution of 6-(carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid (100 mg, 0.40 mmol), (2-amino-4-thiophen-2-yl-phenyl)-carbamic acid tert-butyl ester (172 mg, 0.59 mmol) and N,N-diisopropylethylamine (0.103 mL, 0.59 mmol) in DMF (2.0 mL) was added (1H-1,2,3-benzotriazol-1-yloxy)(triisopropyl)phosphonium hexafluorophosphate (262 mg, 0.59 mmol) and the reaction was stirred overnight. The solvent was removed and the residue was purified by reverse phase chromatography (10-100% ACN/H2O). The fractions were extracted with EtOAc, washed with brine, dried over MgSO4 and concentrated in vacuo. ESIMS calcd 526 (M++H), found 526 (M++H).

[0297]Step 2: TFA Deprotection;

[0298]To a solution of (2-{[6-(carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carbonyl]-amino}-4-thiophen-2-yl-phenyl)-carbamic acid tert-butyl ester (163 mg, 0.31 mmol)) in methylene chloride (2 mL) was added trifluoroacetic acid (1 mL) and the solution was stirred at ambient temperature for 16 hours. The reaction was evaporated to dryness. The residue was washed with sat. NaHCO3 and MeOH, and the solid was filtered. 1H NMR (DMSO-d6) δ 10.03 (br s, 1H), 8.33 (s, 1H), 8.11 (s, 1H), 8.02 (d, J=8.2 Hz, 1H), 7.93 (s, 1H), 7.62 (br d, J=2.4 Hz, 1H), 7.51 (d, J=8.2 Hz, 1H), 7.45 (d, J=1.8 Hz, 1H), 7.33 (dd, J=5.0, 1.8 Hz, 1H), 7.29 (dd, J=8.2, 1.8 Hz, 1H), 7.23 (dd, J=3.5, 1.8 Hz, 1H), 7.02 (dd, J=5.2, 3.5 Hz, 1H), 6.79 (d, J=8.2 Hz, 1H), 5.93 (d, J=45.7 Hz, 1H), 5.24 (s, 2H). cal'd 427 (MH+), exp 427 (MH+).

6-[(4-Chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide

[0299]Step 1: Coupling; 6-[(4-Chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid ethyl ester. A solution of 6-(carboxy-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester (180 mg, 1.42 mmol), EDCI (134 mg, 0.70 mmol), HOBt (95 mg, 0.70 mmol) and 4-chlorobenzylamine (0.94 mL, 0.77 mmol) in DMF (4 mL) was stirred overnight. The solvent was removed. The residue was diluted with EtOAc, washed with H2O, and NaHCO3. The combined organic layers were dried over Na2SO4, filtered, concentrated yielded the crude amide, which was used without further purification. cal'd 406 (MH+), exp 406 (MH+).

[0300]Step 2: Hydrolysis; 6-[(4-Chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid. To a solution of 6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid ethyl ester (241 mg, 0.60 mmol) in MeOH/THF (⅔ mL) was added 2M NaOH (0.65 mL). The resultant solution was stirred overnight and 2N HCl was added dropwise to acidify (˜0.7 mL). The solvent was removed in vacuo and the solid was used without further purification. cal'd 378 (MH+), exp 378 (MH+).

[0301]Step 3: Coupling; [3-({6-[(4-Chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carbonyl}-amino)-biphenyl-4-yl]-carbamic acid tert-butyl ester. A solution of 6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (108 mg, 0.286 mmol), EDCI (74 mg, 0.383 mmol), HOBt (52 mg, 0.383 mmol) and (3-amino-biphenyl-4-yl)-carbamic acid tert-butyl ester (87 mg, 0.306 mmol) in DMF (4 mL) was stirred overnight. The solvent was removed. The residue was diluted with EtOAc, washed with H2O, and NaHCO3. The combined organic layers were dried over Na2SO4, filtered, concentrated yielded the crude amide, which was used without farther purification. cal'd 378 (MH+), exp 378 (MH+).

[0302]Step 4: TFA Deprotection; 6-[(4-Chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide. To a solution of [3-({6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carbonyl}-amino)-biphenyl-4-yl]-carbamic acid tert-butyl ester (164 mg, 0.26 mmol)) in methylene chloride (2 mL) was added trifluoroacetic acid (1 mL) and the solution was stirred at ambient temperature for 16 hours. The reaction was evaporated to dryness. The residue was washed with sat. NaHCO3 and MeOH, and the solid was filtered. 1H NMR (DMSO-d6) δ 10-05 (br s, 1H), 9.12 (t, J=5.0 Hz, 1H), 8.08 (br s, 1H), 8.00 (m, 1H), 7.55-7-51 (m, 3H), 7.49-7.45 (m, 1H), 7.38-7.33 (m, 5H), 7.33-7.27 (m, 1H), 7.25 (d, J=8.4 Hz, 2H), 7.21 (t, J=6.9 Hz, 1H), 6.85-6.80 (m, 1H), 6.07 (d, J=45.7 Hz, 1H), 5.15 (s, 2H), 4,334.26 (m, 2H). cal'd 545 (MH+), exp 545 (MH+).

Compounds from 5- and 6-azidomethylbenzothiophenes.

[0303]N-(2-aminophenyl)-6(azidomethyl)-1-benzothiophene-2-carboxamide. 6-Hydroxymethyl-benzo[b]thiophene-2-carboxylic acid ethyl ester (500 mg, 2.12 mmol) was dissolved in CH2Cl2 (10 mL) and Et3N (443 mL, 3.18 mmol) followed by mesyl chloride (197 mL, 2-54 mmol) were added. The solution was allowed to stir for 30 min and the solution was poured into 50 mL of Et2O— The organic solution was washed with 50 mL of 1 M KHSO4 followed by 50 mL of brine. The solution was dried over MgSO4 and the crude mesylate was used without further purification. This mesylate was dissolved in DMF (5 mL) and NaN3 (325 mg, 5.00 mmol) was added. The suspension was allowed to stir for 30 min then poured into 50 mL of water and 50 mL of EtOAc. The layers were separated then the organic solution was washed with 50 mL of brine and dried over MgSO4. The solution was concentrated to give the crude azide, which was used without further purification. The azide was dissolved in 8.80 mL of THF and 2.20 mL of MeOH and 2.20 mL of a 1 M solution of LiOH (2.20 mmol) was added. The solution was allowed to stir for 18 h, then diluted with 50 mL of EtOAc and washed with 50 mL of 1M KHSO4 followed by 50 mL of brine. The organic layer was dried and concentrated to give the crude acid that was used without further purification. This acid was dissolved in 4 mL of DMF. 1,2-phenylenediamine (459 mg, 4.24 mmol), EDC (810 mg, 4.24 mmol), and HOBT (573 mg, 4.24 mmol) were added and the solution was allowed to stir for 2 h. The solution was diluted with 50 mL of EtOAc and washed with 50 mL of water and 50 mL of brine. The organic solution was dried and concentrated then purified by flash chromatography (12-100% EtOAc in hexanes) to give the amide as a light yellow powder. 1H NMR (600 MHz, CDCl3) δ 4.49 (s, 2), 6.85 (t, 2, J=6.1), 7.10 (t, 1, J=7.4), 7.37 (t, 2, J=8.5), 7.82 (s, 1), 7.85 (d, 1, J=8.2), 7.90 (m, 2). MS cal'd 324 (MH+), exp 324 (MH+).

[0304]N-(2-aminophenyl)-6-{[4-(2-phenylethyl)1H-1,2,3-triazol-1-yl]methyl}-1-benzothiophene-2-carboxamide. CuSO4 (16 μL of 7.5% w/v sol'n, 0.0075 mmol) was added to a solution of N-(2-aminophenyl)-6-(azidomethyl)-1-benzothiophene-2-carboxamide (32 mg, 0.10 mmol) and 4-phenyl-1-butyne (16.8 μL, 0.120 mmol) in MeOH. Sodium ascorbate (20 μL of a 1 M sol'n, 0.020 mmol) was added followed by Ph3P (5.9 mg, 0.023 mmol). The solution was allowed to stir overnight then the solution was directly purified by reverse phase HPLC to give the trazole as a light yellow powder. 1H NMR (600 MHz, CDCl3) δ 2.95-2.97 (m, 2), 3.00-3.01 (m, 2), 5.58 (s, 2), 6.84-6.86 (m, 2), 7.06 (s, 1), 7.10-7.12 (m, 3) 7.15 (t, 1, J=7.4), 7.20-7.23 (m, 3), 7.39 (d, 1, J=7.0), 7.66 (s, 1), 7.80 (d, 1, J=8.2), 7.88 (s, 1), 7.95 (s, 1). MS cal'd 454 (MH+), exp 454 (MH+).

Synthesis of Diaminoarylpyrazoles.

[0305]

tert-butyl (3-amino-1-phenyl-1H-pyrazol-4-yl)carbamate

Step A: Copper Coupling

[0306]A solution of methyl 4-nitro-1H-pyrazole-3-carboxylate (54.0 g, 315.6 mmol), phenylboronic acid (77.0 g, 631.2 mmol), copper(II) acetate (86.0 g, 473.4 mmol) and pyridine (49.9 g, 631.2 mmol) in methylene chloride (600 mL) was stirred at ambient temperature open to air for 48 hours. The reaction was evaporated in vacuo, diluted with 1000 mL methylene chloride and filtered through a large plug of silica (washing with 2 liters methylene chloride). The solvent was evaporated in vacuo. 1H NMR (CDCl3) i 8.61 (s, 1H), 7.73 (m, 2H), 7.50 (m, 3H), 4.02 (s, 3H).

Step B: Saponification

[0307]A solution of methyl 4-nitro-1-phenyl-1H-pyrazole-3-carboxylate (78.1 g, 315.9 mmol) in THF (600 mL) was treated with 4M potassium hydroxide (79 mL, 316 mmol) dropwise and the solution was stirred at ambient temperature for 16 hours. The reaction was evaporated in vacuo and acidified with 6M HCl. After addition of water (500 mL) the solids were filtered off and dried to give 72.1 g (97%, 2 steps) of desired compound as a grayish solid. 1H NMR (CD3OD) δ 9.37 (bs, 1H), 7.88 (m, 2H), 7.59 (m, 2H), 7.44 (m, 1H).

Step C: Curtius

[0308]A solution of 4-nitro-1-phenyl-1H-pyrazole-3-carboxylic acid (20.0 g, 85.8 mmol), triethylamine (36.0 mL, 257.3 mmol), and diphenylphosphoryl azide (37.8 g, 137.2 mmol) in dioxane (400 mL) and tert-butanol (200 mL) was heated to reflux for 16 hours. The reaction was evaporated to dryness in vacuo, diluted with methylene chloride (400 mL) and treated with trifluoroacetic acid (128 g, 857.7 mmol). The solution was stirred at ambient temperature for 16 hours. The reaction was evaporated in vacuo and the resulting oil diluted with hexanes (750 mL), ethyl acetate (150 mL) and methylene chloride (100 mL). The solids were filtered, washed with above solvent system (hexanes:ethyl acetate; methylene chloride 75:15:10), and dried to give 12.0 g of desired product as yellow solid. 1H NMR (CDCl3) δ 8.43 (s, 1H), 7.62 (m, 2H), 7.48 (m, 2H), 7.37 (m, 1H).

Step D: Hydrogenation/Boc Protection

[0309]A solution of 4-nitro-1-phenyl-1H-pyrazol-3-amine (0.15 g, 0.74 mmol), di-tertbutyl dicarbonate (0.16 g, 0.74 mmol), triethylamine (0.19 g, 1.84 mmol) in methanol 20 mL was degassed with nitrogen and treated with platinum oxide (17 mg, 10 mol %). The solution was placed under a hydrogen atmosphere and stirred at ambient temperature for 2 hours. The reaction was then degassed with nitrogen, filtered through celite, washed with methanol and evaporated in vacuo Flash chromatography (20-35% ethyl acetate/hexanes) gave 0.109 g (54%) of title compound as a purplish solid. 1H NMR (CDCl3) δ 7.85 (s, 1H), 7.51 (m, 2H), 7.37 (m, 2H), 7.18 (m, 1H), 6.40 (bs, 1H).

General Procedure for Coupling of Diaminoarylpyrazoles.

[0310]

N-(4-amino-1-phenyl-1H-pyrazol-3-yl)-6-[2-(benzylamino)-2-oxoethyl]-1-benzothiophene-2-carboxamide

Step A: Bop Coupling

[0311]To a solution of 6-[2-(benzylamino)-2-oxoethyl]-1-benzothiophene-2-carboxylic acid (0.25 g, 0.77 mmol), tert-butyl (3-amino-1-phenyl-1H-pyrazol-4-yl)carbamate (0.26 g, 0.96 mmol) and N,N-diisopropylethylamine (0.15 g, 1.15 mmol) in methylene chloride (5 mL) was added (1H-1,2,3-benzotriazol-1-yloxy)(triisopropyl)phosphonium hexafluorophosphate (0.51 g, 1.15 mmol). The reaction was sealed and heated to 60° C. for 16 hours. The reaction was purified by flash chromatography (0-2.5% methanol/methylene chloride) to give crude product. ESIMS calcd 582.2 (M++H), found 582.2 (m++H).

Step B: Deprotection

[0312]To a solution of crude tert-butyl {3-[({6-[2-(benzylamino)-2-oxoethyl]-1-benzothien-2-yl}carbonyl)amino]-1-phenyl-1H-pyrazol-4-yl}carbamate (from step A) in ethyl acetate (10 mL) and methanol (10 mL) was added 4M HCl in dioxane (10 mL) and the resulting solution was stirred for 16 hours at ambient temperature. The reaction was evaporated to dryness and purified by reverse phase LC to give 148 mg (40%, 2 steps) of N-(4-amino-1-phenyl-1H-pyrazol-3-yl)-6-[2-(benzylamino)-2-oxoethyl]-1-benzothiophene-2-carboxamide as a white solid. 1H NMR (DMSO-d6) δ 11.01 (s, 1H), 8.59 (t, J=5.87 Hz, 1H), 8.35 (s, 1H), 7.89 (t, J=3.82 Hz, 2H), 7.80 (s, 1H), 7.67 (d, J=7.92 Hz, 2H), 7.42 (t, J=7.33 Hz, 2H), 7.36 (d, J=9.1 Hz, 1H), 7.28 (t, J=7.92 Hz, 2H), 7.19 (m, 4H), 4.25 (d, J=5.87 Hz, 2H), 3.61 (s, 2H). ESIMS calcd 482.2 (M++H), found 482.1 (M++H).

[0313]Additional α-aminoaryl analogs were prepared in procedures similar to those described for the preparations of the above examples. Unless otherwise indicated, the compounds were isolated as the free form (parent).

1 Cpd # Ra Name MS 1-1 6-(Benzylcarbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 510 (MH+),510 (MH+) 1-2 6-(Fluoro-phenylcarbamoyl-methyl)-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 496 (MH+),exp 496 (H+) 1-3 6-[(4-Chloro-phenylcarbamoyl)-fluoro-methyl]-benzyl[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 531 (MH+),exp 531 (MH+) 1-4 6-[(4-Chloro-benzylcarbamoyl)-fluorro-methyl]-benzo[b]thiophene-2-carboxylicacid (4-amino-biphenyl-3-yl)amide cal'd 545 (MH+),exp 545 (MH+) 1-5 6-{[1-(S)-(4-Chloro-phenyl)-ethylcarbamoyl]-fluoro-methyl}-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 558, 560(MH+), 558, 560(MH+) 1-6 6-[(2,4-Dichloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 579 (MH+),exp 579 (MH+) 1-7 6-(Carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylicacid (4-amino-biphenyl-3-yl)amide cal'd 420 (MH+),exp 420 (MH+) 1-8 6-(Fluoro-methylcarbamoyl-methyl)-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 434 (MH+),exp 434 (MH+) 1-9 6-[(4-Amino-biphenyl-3-ylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylicacid (4-amino-biphenyl-3-yl)amide cal'd 587 (MH+),exp 587 (MH+) 1-10 6-[(2,2-Difluoro-1-phenyl-ethylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)ammide cal'd 560 (MH+),exp 560 (MH+) 1-11 6-{[1-(R)-(4-Chloro-phenyl)-ethylcarbamoyl]-fluoro-methyl}-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide cal'd 558, 560(MH+), 558, 560(MH+) 1-12 6-[Fluoro-(indan-1-(S)-ylcarbamoyl)-methyl]-benzo[b]thiophene-2-carboxylicacid (4-amino-biphenyl-3-yl)amide cal'd 536 (MH+),536 (MH+) 1-13 6-{[1-(S)-Phenyl-ethylcarbamoyl]-fluoro-methyl}-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide; isolated asthe TFA salt cal'd 524 (MH+),524 (MH+)

2 Cpd # Ra Name MS 2-1 6-(Benzylcarbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-2-yl-phenyl)amide cal'd 516 (MH+),516 (MH+) 2-2 6-(Carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylicacid (2-amino-5-thiophen-2-yl-phenyl)amide cal'd 426 (MH+),exp 426 (MH+) 2-3 6-(Fluoro-methylcarbamoyl-methyl)-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-2-yl-phenyl)amide cal'd 440 (MH+),exp 440 (MH+) 2-4 6-[Fluoro(1-(S)-phenyl-ethylcarbamoyl)-methyl]-benzo[b]thiophene-2-carboxylicacid (2-amino-5-thiophen-2-yl-phenyl)amide cal'd 530 (MH+),530 (MH+) 2-5 6-[(2-Amino-5-thiophen-2-yl-phenylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-2-yl-phenyl)amide cal'd 599 (MH+),exp 599 (MH+)

3 Cpd # Ra Name MS 3-1 6-(Benzylcarbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-3-yl-phenyl)ammide;isolated as the TFA salt cal'd 516 (MH+),516 (MH+)

[0314]Additional α-aminopyrazole analogs were prepared in procedures similar to those described for the preparation of the above N-(4-amino-1-phenyl-1H-pyrazol-3-yl)-6-[2-(benzylamino)-2-oxoethyl]-1-benzothiophene-2-carboxamide.

4 Cpd # Ra Rb Name MS 4-1 3-Cl 6-(Benzylcarbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid [4-amino-1-(3-chloro-phenyl)-1H-pyrazol-3-yl]-amide; isolated as the TFAsalt cal'd 534.1(MH+),534.0 (MH+) 4-2 3-Cl 6-(Benzylcarbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylic acid [4-amino-1-(3-chloro-phenyl)-1H-pyrazol-3-yl]-ammide cal'd 534.1(MH+), exp534.1 (MH+) 4-3 H 6-(Carbamoyl-fluoro-methyl)-benzo[b]thiophene-2-carboxylicacid (4-amino-1-phenyl-1H-pyrazol-3-yl)amide cal'd 410 (MH+),exp 410 (MH+) 4-4 H 6-{Fluoro-[(pyridin-3-ylmethyl)-carbamoyl]-methyl}-benzo[b]thiophene-2-carboxylicacid (4-amino-1-phenyl-1H-pyrazol-3-yl)amide; isolated asthe HCl salt cal'd501.1 (MH+), exp501.1 (MH+) 4-5 H N-(4-amino-1-phenyl-1H-pyrazol-3-yl)-6-[2-(benzylamino)-1-fluoro-2-oxoethyl]-1-benzothiophene-2-carboxamide cal'd 500 (MH+),exp 500 (MH+)

Oxadiazoles

[0315]

6-[Fluoro(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]-1-benzothiophene-2-carboxylic acid

[0316]Step A: EDC Coupling; 6-[2-(N′-Benzoyl-hydrazino)-1-fluoro-2-oxo-ethyl]-benzo[b]thiophene-2-carboxylic acid ethyl ester. A solution of [2-(ethoxycarbonyl)-1-benzothien-6-yl](fluoro)acetic acid (50 mg, 0.18 mmol) and benzohydrazide (36.2 mg, 0.27 mmol) in methylene chloride (2 mL) was treated with EDC (51 mg, 0.27 mmol) and the resulting solution was stirred at ambient temperature for 3 hours. The reaction was purified by flash chromatography (1-5% methanol/methylene chloride) to give ethyl 6-[2-(2-benzoylhydrazino)-1-fluoro-2-oxoethyl]-1-benzothiophene-2-carboxylate as a white solid. ESIMS calcd 401.1 (M++H), found 401.0 (M++H).

[0317]Step B: Dehydration/Saponification; 6-[Fluoro-(5-phenyl-[1,3,4]oxadiazol-2-yl)-methyl]-benzo[b]thiophene-2-carboxylic acid. A solution of ethyl 6-[2-(2-benzoylhydrazino)-1-fluoro-2-oxoethyl]-1-benzothiophene-2-carboxylate (51 mg, 0.13 mmol) in THF (2.0 mL) was treated with Burgess reagent (45 mg, 0.19 mmol). The reaction vessel was sealed and heated to 120° C. in the microwave reactor for 20 minutes. The reaction was evaporated in vacuo and purified by flash chromatography (10-40% ethyl acetate/hexanes) to give ethyl 6-[fluoro(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]-1-benzothiophene-2-carboxylate as a white solid. ESIMS calcd 383.1 (M++H), found 383.0 (M++H). To a solution of ethyl 6-[fluoro(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]-1-benzothiophene-2-carboxylate in THF (2.5 mL) was added 1M sodium hydroxide (5.0 mL, 5.0 mmol) and the resulting solution was stirred at ambient temperature for 1 hour. The reaction was partitioned between ethyl acetate and 1M HCl solution. The organics were dried over sodium sulfate, filtered and evaporated to give 6-[fluoro(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]-1-benzothiophene-2-carboxylic acid as a white solid. ESIMS calcd 355.1 (M++H), found 355.0 (M++H).

[0318]α-aminoaryl analogs from the carboxylic acids were prepared in procedures similar to those described for the preparations of the above examples. All of the compounds were isolated as the TFA salt.

5 Cpd # Ra Name MS 5-1 Me 6-[Fluoro-(5-methyl- cal'd 449.1 [1,3,4]oxadiazol-2-yl)-methyl]- (MH+), exp benzo[b]thiophene-2-carboxylic acid 449.1 (MH+) (4-amino-1-phenyl-1H-pyrazol-3- yl)amide 5-2 6-{Fluoro-[5-(2-methoxy-phenyl)-[1,3,4]oxadiazol-2-yl]-methyl}-benzo[b]thiophene-2-carboxylic acid(4-amino-1-phenyl-1H-pyrazol-3-yl)amide cal'd 541.1(MH+), 541.1(MH+) 5-3 6-[Fluoro-(5-phenyl-[1,3,4]oxadiazol-2-yl)-methyl]-benzo[b]thiophene-2-carboxylic acid(4-amino-1-phenyl-1H-pyrazol-3-yl)amide cal'd 511.1(MH+), 511.1(MH+)

Procedures for A2. Compounds from (carboxy-difluoro-methyl)benzothiophenes.

[0319]3-Formyl-4-nitro-benzoic acid methyl ester. A solution of 3-methyl-4-nitro-benzoic acid methyl ester (24.99 g, 128.1 mmol) and N,N-dimethylformamide dimethyl acetal (40.0 mL, 300 mmol) was heated at 140° C. for 22.5 h. After cooling to rt, the reaction mixture was concentrated and the residue was crystallized from MeOH to give a purple solid. This solid was dissolved in THF (500 mL) and water (500 mL), and sodium periodate (62.62 g, 292.8 mmol) was added followed by additional sodium periodate (15.6 g, 72.9 mmol) two hours later. After stirring at rt for an additional 1 h, the reaction mixture was filtered through Celite washing with EtOAc (2 L). The filtrate was washed with saturated NaHCO3 (600 mL) and the organic layer was dried over Na2SO4. After filtration, the filtrate was concentrated and the residue was passed through a pad of silica gel, washing with CH2Cl2/hexanes (75%-100%). The filtrate was concentrated and dried to give 3-formyl-4-nitro-benzoic acid methyl ester as yellowish solid. MS (EI): cal'd 210.0 (MH+), exp 210.2 (M+).

[0320]Benzo[b]thiophene-2,6-dicarboxylic acid 2-ethyl ester 6-methyl ester. A mixture of 4-formyl-3-nitro-benzoic acid methyl ester (15.2 g, 72.8 mmol), mercapto-acetic acid ethyl ester (8.70 mL, 79.3 mmol) and K2CO3 (12.9 g, 93.1 mmol) in 140 mL of anhydrous DMF was heated at 50° C. overnight. After cooling to rt, the mixture was poured into 1 L of ice-water and the resulting mixture was stirred for 40 min. The solid formed was filtered and washed with 4×70 mL of water. After drying, benzo[b]thiophene-2,6-dicarboxylic acid 2-ethyl ester 6-methyl ester was obtained as a pale solid. 1H NMR (CDCl3, 200 MHz) δ 8.56 (s, 1H), 8.09-7.97 (m, 2H), 7.88 (d, J=8.0 Hz, 1H), 4.40 (q, J=7.2 Hz, 2H), 3.95 (s, 3H), 1.40 (t, J=6.8 Hz, 3H). MS (EI): cal'd 265.0 (MH+), exp 265.0 (MH+).

[0321]Benzo[b]thiophene-2,6-dicarboxylic acid 2-ethyl ester. A mixture of benzo[b]thiophene-2,6-dicarboxylic acid 2-ethyl ester 6-methyl ester (14.9 g, 56.4 mmol) and LiI (38.0 g, 284 mmol) in 120 mL of anhydrous pyridine was refluxed for 3 h. After cooling to rt, the mixture was poured into ice-cold 2N HCl (800 mL). The solid formed was filtered and washed with 3×100 mL of water. After drying, the solid was crystallized from MeOH to give benzo[b]thiophene-2,6-dicarboxylic acid 2-ethyl ester as a pale solid. 1H NMR (DMSO-d6, 200 MHz) δ 8.66 (s, 1H), 8.21 (s, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.96 (dd, J=8.4, 1.0 Hz, 1H), 4.34 (q, J=7.2 Hz, 2H), 1.40 (t, J=6.8 Hz, 3H). MS (EI): cal'd 251.0), exp 251.1 (MH+).

[0322]Ethyl 6{[methoxy(methyl)amino]carbonyl}-1-benzothiophene-2-carboxylate. 2-(ethoxycarbonyl)-1-benzothiophene-6-carboxylic acid (2.5 g, 10.0 mmol) was dissolved in DMF (20 mL) and cooled to 0° C. EDCI (1.92 g, 10.0 mmol) was added to the reaction followed by (MeO)NHMe.HCl (1.5 g, 15.0 mmol) and then Et3N (1.4 mL, 10.0 mmol). The reaction was allowed to stir for 1 h at 0° C. Water was added to reaction mixture and then extracted with Et2O (3×). The combined organic layers were dried over Na2SO4, filtered, concentrated yielded the crude amide. Purification by flash column chromatography provided the desired amide. 1H NMR (CDCl3, 600 MHz) δ 8.20 (s, 1H), 8.04 (s, 1H), 7.87 (d, J=8.4 Hz, 1H), 7.69 (dd, J=8.4, 1.0 Hz, 1H), 4.39 (d, J=7.2 Hz, 2H), 3.54 (s, 3H), 3.38 (s, 3H), 1.4 (t, J=7.2 Hz, 3H). MS: cal'd (MH+) 294, exp (MH+) 294.

[0323]Ethyl 6-acetyl-1-benzothiophene-2-carboxylate. To a solution of ethyl 6-{[methoxy(methyl)amino]carbonyl}-1-benzothiophene-2-carboxylate (0.07 g, 0.22 mmol) in THF (3 mL) at −78° C. was added MeLi (0.15 mL, 1.6 M in Et2O, 0.24 mmol). The reaction was stirred for 1 h at −78° C. before saturated ammonium chloride solution was added to quench the reaction. At which time the reaction mixture was allowed to warm to room temperature and then extracted with a mixture of hexane:ethyl acetate solution (3:1) (3×). The combined organic layers was dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture was chromatographed to provide the ketone. 1H NMR (CDCl3, 600 MHz) δ 8.46 (s, 1H), 8.07 (s, 1H), 7.98 (dd, J=8.2, 1.2 Hz, 1H), 7.92 (m, 1H), 4.42 (q, J=7.1 Hz, 2H), 2.69 (s, 3H), 1.42 (t, J=7.2 Hz, 3H). MS: cal'd (MH+) 249, exp (MH+) 249.

[0324]6-Carboxyoxalyl-benzo[b]thiophene-2-carboxylic acid ethyl ester. To solution of ethyl 6-acetyl-1-benzothiophene-2-carboxylate (1.0 g, 40.3 mmol) in pyridine (4 mL) at 90° C. was added selenium dioxide (782 mg, 7.05 mmol) portionwise over 1.5 h. After 7 h, cooled to RT and filtered via Celite. The solvent was removed in vacuo and the residue was diluted with EtOAc, washed with 0.5N HCl, dried (MgSO4) and the solvent was evaporated under reduced pressure. The material was used without further purification. 1H NMR (DMSO-d6) δ 8.45 (br s, 1H), 8.22 (br s, 1H), 8.05 (d, J=8.2 Hz, 1H), 7.84 (dd, J=8.2, 1.5 Hz, 1H), 4.34 (q, J=7.3 Hz, 2H), 1.32 (t, J=7.3 Hz, 3H). cal'd 279 (MH+), exp 279 (MH+).

[0325]6-Methoxyoxalyl-benzo[b]thiophene-2-carboxylic acid ethyl ester. To a solution of 6-oxalyl-benzo[b]thiophene-2-carboxylic acid ethyl ester (500 mg, 1.80 mmol) and triethylamine (0.250 mL, 1.80 mmol) in CH2Cl2 (5 ml) at RT was added methyl chloroformate (0.139 ml, 1.80 mmol). After 30 min, the reaction mixture was diluted with CH2Cl2 (5 ml) and washed with H2O. The combined organic extracts were washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Biotage 25M), eluting with EtOAc/hexane to give a yellow solid 1H NMR (DMSO-d6) δ 8.59 (br s, 1H), 8.09 (br s, 1H), 8.03 (dd, J=8.2, 1.5 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H), 4.42 (q, J=7.3 Hz, 2H), 4.02 (s, 3H), 1.41 (t, J=7.3 Hz, 3H). cal'd 293 (MH+), exp 293 (MH+).

[0326]6-(Difluoro-methoxycarbonyyl-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester. To a solution of 6-methoxyoxalyl-benzo[b]thiophene-2-carboxylic acid ethyl ester (445 mg, 1.52 mmol) in CH2Cl2 (15 mL) at RT was added diethylaminosulfur trifluoride (0.453 mL, 3.43 mmol). After 18 h, LC/MS reveals ˜10% starting material, so an additional DAST (0.250 mL) was added. This was repeated every 2 h until the disappearance of the starting material. The reaction mixture was quenched with MeOH (1 mL). The solution was diluted with CH2Cl2 (25 mL) and washed with H2O and brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Biotage 25M), eluting with EtOAc/hexane to give a colorless solid. 1H NMR (DMSO-d6) δ 8.13 (br s, 1H), 8.07 (br s, 1H), 7.94 (br d, J=8.2 Hz, 1H), 7.61 (br d, J=8.2 Hz, 1H), 4.42 (q, J=7.3 Hz, 2H), 3.86 (s, 3H), 1.421 (t, J=7.3 Hz, 31H). cal'd 315 (MH+), exp 315 (MH+).

[0327]6-(Carboxy-difluoro-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester. To a solution of 6-(difluoro-methoxycarbonyl-methyl)-benzo[b]thiophene-2-carboxylic acid ethyl ester (200 mg, 0.64 mmol) in MeOH/THF (½ mL) was added 1M LiOH (0.67 mL). The resultant solution was stirred overnight and 2N HCl was added dropwise to acidify (˜0.7 mL). The solvent was removed in vacuo and the solid was used without further purification. cal'd 301 (MH+), exp 301).

[0328]α-aminoaryl analogs were prepared in procedures similar to those described for the preparation of the above 6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide. The compounds were isolated as the free form (parent).

6 Cpd # Ra Name MS 6-1 6-(Diethylcarbamoyl-difluoro-methyl)-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-2-yl-phenyl)amide cal'd 500(MH+), exp500 (MH+) 6-2 6-(Carbamoyl-difluoro-methyl)-benzo[b]thiophene-2-carboxylicacid (2-amino-5-thiophene-2-yl-phenyl)amide cal'd 444(MH+), exp444 (MH+)

7 Cpd # Ra Name MS 7-1 6-(Diethylcarbamoyl-difluoro-vmethyl)-benzo[b]thiophene-2-carboxylic acid (2-amino-5-thiophen-3-yl-phenyl)amide cal'd 500(MH+), exp500 (MH+)

Procedures for B1. Compounds from (carboxy-fluoro-methyl)-benzoic acids.

[0329]2-(4-tert-Butoxycarbonyl-phenyl)-malonic acid dimethyl ester To a mixture of tert-butyl 4-bromobenzoate (10.1 g, 39.2 mmol), K3PO4 (20.2 g, 95.0 mmol), and dimethyl malonate (4.50 mL, 39.2 mmol) was added toluene (69 mL), Pd2(dba)3 (975 mg, 1.08 mmol), and P(tert-Bu)3 (12.8 mL, 10% wt. in hexanes, 4.3 mmol). The reaction mixture was degassed and heated to 85° C. After 2 d, the reaction mixture was diluted with ethyl acetate, washed with water (1×), brine (1×), dried over MgSO4, filtered, and concentrated. Purification by flash column chromatography on silica gel (5% to 20% ethyl acetate in hexanes) gave dimethyl [4-(tert-butoxycarbonyl)phenyl]malonate as a low melting white solid: 1H NMR (600 MHz, CDCl3) δ 7.98 (d, J=8.5 Hz, 2H), 7.45 (d, J=8.2 Hz, 2H), 4.70 (s, 1H), 3.76 (s, 6H), 1.58 (s, 9H); ESIMS calcd 331.1 (M++Na), found 331.1 (M++Na).

[0330]2-(4-tert-Butoxycarbonyl-phenyl)-2-fluoro-malonic acid dimethyl ester. To a solution of dimethyl [4-(tert-butoxycarbonyl)phenyl]malonate (1.0 g, 3.24 mmol) in THF (10 mL) at 0° C. was added sodium hydride (014 g, 3.6 mmol, 60% dispersion in mineral oil). The reaction was stirred at 0° C. for 15 minutes. A solution of Selectfluor (1.26 g, 3.6 mmol) in DMF (10 mL) was added and the reaction was allowed to warm to ambient temperature and stir under nitrogen for 1 hour. The reaction was quenched with ammonium chloride solution and partitioned between water/ethyl acetate. The organics were dried over sodium sulfite, filtered and evaporate. Purification by flash chromatography (0-20% ethyl acetate/hexanes) gave the desired product as a white solid. 1H NMR (600 MHz, CDCl3) δ 8.02 (d, J=8.2 Hz, 2H), 7.65 (d, J=8.5 Hz, 2H), 3.86 (s, 3H), 1.58 (s, 9H); ESIMS calcd 349.1 (M++Na), found 349.1 (M++Na).

[0331]2-(4-tert-Butoxycarbonyl-phenyl)-2-fluoro-malonic acid diethyl ester. A stirred slurry of o-fluoro-diethyl malonate (6.93 g, 38.9 mmol), 4-bromo-benzoic acid tert-butyl ester (10 g, 38.9 mmol) and potassium phosphate (21.5 g, 101 mmol) was degassed, then bis(tri-t-butylphosphine)palladium(0) (0.994 g, 1.95 mmol) was added. The resulting mixture was degassed again before stirring at 85° C. under N2 over the weekend. The mixture was cooled, diluted with EtOAc (100 mL), washed with water, dried (MgSO4), filtered and the solvent was evaporated under reduced pressure. Purification of the residue by MPLC using 2-20% EtOAc/Hexanes gave 2-(4-tert-butoxycarbonyl-phenyl)-2-fluoro-malonic acid diethyl ester. 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J=8.2 Hz, 2H), 7.64 (d, J=8.2 Hz, 2H), 4.31 (q, J=7-5 Hz, 4H), 1.29 (t, J=7.5 Hz, 6H); cal'd 355 (MH+), exp 355 (MH+).

[0332]4-(Carboxy-fluoro-methyl)-benzoic acid tert-butyl ester. A stirred solution of 2-(4-tert-butoxycarbonyl-phenyl)-2-fluoro-malonic acid diethyl ester (2.97 g, 8.38 mmol) in MeOH/THF ( 5/15 mL) was added 2M NaOH (12.6 mL) and then the mixture was degassed. After 18 h, the solution was heated to 60° C. for 30 min. The mixture was cooled, diluted with EtOAc (100 mL), washed with water, and 0.5 N citric acid, then dried (MgSO4), filtered and the solvent was evaporated under reduced pressure. To the residue was added EtOAc and H2O, and the mixture was heated to 100° C. for 1 h. The mixture was cooled, diluted with EtOAc (100 mL), washed with water, and 0.5 N citric acid, then dried (MgSO4), filtered and the solvent was evaporated under reduced pressure. The material was used w/o further purification. cal'd 255 (MH+), exp 255 (MH+).

[0333]4-(Fluoro-methylcarbamoyl-methyl)-benzoic acid tert-butyl ester. To a solution of 4-(carboxy-fluoro-methyl)-benzoic acid tert-butyl ester (800 mg, 3.15 mmol), NMM (1.2 eq), and oxalyl chloride (0.33 mL, 3.78 mmol) in CH2Cl2 (20 mL) was added one drop of DMF. The solution was stirred for 1 h, then added to a solution of ammonium hydroxide (1.9 mL, 15.7 mmol) in CH2Cl2 (10 mL). After 30 min, the solvent was removed. The residue was diluted with CH2Cl2 (50 mL), washed with water, then dried (MgSO4), filtered and the solvent was evaporated under reduced pressure. The material was used w/o further purification. 1H NMR (600 MHz, DMSO-d6) δ 7.91 (d, J=8.2 Hz, 2H), 7.89 (br s, 1H), 7.59 (br s, 1H), 7.53 (d, J=8.2 Hz, 2H), 5.87 (d, J=47.8 Hz, 1H), 1.52 (s, 9H); cal'd 254 (MH+), exp 254 (MH+).

[0334]4-(Carbamoyl-fluoro-methyl)-benzoic acid. To a solution 4-(fluoro-methylcarbamoyl-methyl)-benzoic acid tert-butyl ester (309 mg, 1.22 mmol)) in methylene chloride (2 mL) was added trifluoroacetic acid (1 mL) and the solution was stirred at ambient temperature for 4 hours. The reaction was evaporated to dryness. The material was used w/o further purification. 1H NMR (600 MHz, DMSO-d6) δ 13.17 (s, 1H), 7.96 (d, J=8.2 Hz, 2H), 7.85 (br s, 1H), 7.60 (br s, 1H), 7.53 (d, J=8.2 Hz, 2H), 5.88 (d, J=47.8 Hz, 1H); cal'd 198 (MH+), exp 198 (MH+).

[0335]α-aminoaryl analogs were prepared in procedures similar to those described for the preparation of the above 6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide. Optically pure compounds were prepared via separation of Boc-protected intermediates using chiral chromatography and subsequent individual deprotection. All compounds were prepared as the free base (parent) form.

8 Cpd # Ra Name MS 8-1 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(carbamoyl-fluoro-methyl)-benzamide cal'd 370 (MH+),exp 370 (MH+) 8-2 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(dimethylcarbamoyl-fluoro-methyl)-benzamide cal'd 398 (MH+),exp 398 (MH+) 8-3 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(fluoro-isopropylcarbamoyl-methyl)-benzamide cal'd 412 (MH+),exp 412 (MH+) 8-4 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(1-fluoro-2-oxo-2-pyrrolidin-1-yl-ethyl)-benzamide cal'd 424 (MH+),exp 424 (MH+) 8-5 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(1-fluoro-2-oxo-2-pyrrolidin-1-yl-ethyl)-benzamide cal'd 424 (MH+),exp 424 (MH+) 8-6 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(fluoro-methylcarbamoyl-methyl)-benzamide cal'd 384 (MH+),exp 384 (MH+) 8-7 [4-(2-Amino-5-thiophen-2-yl-phenylcarbamoyl)-phenyl]-fluoro-acetic acid methyl ester cal'd 385 (MH+),exp 385 (MH+) 8-8 [4-(2-Amino-5-thiophen-2-yl-phenylcarbamoyl)-phenyl]-fluoro-acetic acid cal'd 371 (MH+),exp 371 (MH+) 8-9 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-[1-fluoro-2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-benzamide cal'd 453 (MH+),exp 453 (MH+) 8-10 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(1-fluoro-2-oxo-2-piperazin-1-yl-ethyl)-benzamide cal'd 439 (MH+),exp 439 (MH+) 8-11 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(1-fluoro-2-morpholin-4-yl-2-oxo-ethyl)-benzamide cal'd 440 (MH+),exp 440 (MH+) 8-12 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-[2-(3-dimethylamino-pyrrolidin-1-yl)-1-fluoro-2-oxo-ethyl]-benzamide cal'd 467 (MH+),exp 467 (MH+) 8-13 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-((S)-fluoro-methylcarbamoyl-methyl)-benzamide cal'd 384 (MH+),exp 384 (MH+) 8-14 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-((R)-fluoro-methylcarbamoyl-methyl)-benzamide cal'd 384 (MH+),exp 384 (MH+) 8-15 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-[(1R)-fluoro-2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-benzamide cal'd 453 (MH+),exp 453 (MH+) 8-16 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-[(1S)-fluoro-2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-benzamide cal'd 453 (MH+),exp 453 (MH+) 8-17 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(carbamoyl-(S)-fluoro-methyl)-benzamide cal'd 370 (MH+),exp 370 (MH+) 8-18 N-(2-Amino-5-thiophen-2-yl-phenyl)-4-(carbamoyl-(R)-fluoro-methyl)-benzamide cal'd 370 (MH+),exp 370 (MH+)

9 Cpd # Ra Name MS 9-1 N-(2-Amino-5-thiophen-3-yl-phenyl)-4-(carbamoyl-fluoro-methyl)-benzamide cal'd 370 (MH+),exp 370 (MH+)

10 Cpd # Ra Rb Name MS 10-1 3-CN H N-(4-Amino-1-phenyl- cal'd 455.1 1H-pyrazol-3-yl)-4-[(3- (MH+), exp 455.0 cyano-phenylcarbamoyl)- (MH+) fluoro-methyl]-benzamide

Oxadiazoles

[0336]α-aminoaryl analogs were prepared in procedures similar to those described for the preparation of analogs from 6-[fluoro(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]-1-benzothiophene-2-carboxylic acid. Unless otherwise indicated, the compounds were prepared as the free base (parent) form.

11 Cpd # Ra Rb Name MS 11-1 2-OMe H N-(4-Amino-1-phenyl-1H-pyrazol- cal'd 485.1 3-yl)-4-{fluoro-[5-(2-methoxy- (MH+), exp phenyl)-[1,3,4]oxadiazol-2-yl]- 485.1 (MH+) methyl}-benzamide; isolated as the TFA salt 11-2 2-OMe F N-(4-Amino-5-fluoro-1-phenyl-1H- cal'd 503.1 pyrazol-3-yl)-4-{fluoro-[5-(2- (MH+), exp methoxy-phenyl)-[1,3,4]oxadiazol- 503.1 (MH+) 2-yl]-,ethyl}-benzamide

Procedures for B2. Compounds from (carboxy-difluoro-methyl)-benzoic acids.

[0337]4-Oxalyl-benzoic acid. To a solution of ethyl (4-cyanophenyl)(oxo)acetate (5.0 g, 24.6 mmol) in water (100 mL) was added concentrated hydrochloric acid (100 mL). The solution was heated to reflux for 16 hours. The solution was cooled to ambient temperature and the solids were filtered and dried to give 4-(carboxycarbonyl)benzoic acid as a white solid. ESIMS calcd 195.0 (M++H), found 195.0 (M++H).

[0338]4-Methoxyoxalyl-benzoic acid methyl ester. To a solution of 4-(carboxycarbonyl)benzoic acid (4.55 g, 23.4 mmol) in methanol (100 mL) was added thionyl chloride (7.0 g, 58.6 mmol) dropwise. The reaction was stirred at ambient temperature for 16 hours then evaporated to dryness to give methyl 4-[methoxy(oxo)acetyl]benzoate as a white solid. 1H NMR (CDCl3) δ 8.15 (m, 2H), 8.09 (m, 2H), 3.99 (s, 3H), 3.96 (s, 3H); ESIMS calcd 223.0 (M++H), found 223.0 (M++H).

[0339]4-(Difluoro-methoxycarbonyl-methyl)-benzoic acid methyl ester. To a solution of methyl 4-[methoxy(oxo)acetyl]benzoate (4.88 g, 22.0 mmol) in methylene chloride (300 mL) was added [bis(2-methoxyethyl)amino]sulfur trifluoride (10.2 g, 46.1 mmol) and the resulting solution was stirred for 16 hours at ambient temperature. The reaction was evaporated to dryness and purified by flash chromatography (0-10% ethyl acetate/hexanes) to give methyl 4-(1,1-difluoro-2-methoxy-2-oxoethyl)benzoate as a white solid. 1H NMR (CDCl3) δ 8.11 (d, J=8.51 Hz, 2H), 7.67 (d, J=8.51 Hz, 2H), 3.93 (s, 3H), 3.85 (s, 3H); ESIMS calcd 245.1 (M++H), found 245.0 (M++H).

[0340]4-(Carboxy-difluoro-methyl)-benzoic acid methyl ester. To a solution of methyl 4-(1,1-difluoro-2-methoxy-2-oxoethyl)benzoate (1.0 g, 4.1 mmol) in THF (20 mL) was added sodium hydroxide (4.1 mmol, 4.1 mL of a 1M solution) dropwise at ambient temperature. The resulting solution was stirred for 16 hours. The reaction was partitioned between 1M HCl and ethyl acetate. The organics were dried over sodium sulfate, filtered and evaporated to give difluoro[4-(methoxycarbonyl)phenyl]acetic acid as a white solid. 1H NMR (CDCl3) δ 8.12 (d, J=8.5 Hz, 2H), 7.71 (d, J=8.21 Hz, 2H), 3.94 (s, 3H), 3.0 (bs, 1H); ESIMS calcd 231.0 (M+H), found 231.0 (M++H).

[0341]α-aminoaryl analogs were prepared in procedures similar to those described for the preparation of the above 6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide. All compounds were prepared as the free base parent) form.

12 Cpd # Ra Name MS 12-1 N-(2-Amino-5-thioiphen-2-yl-phenyl)-4-(difluoro-methylcarbamoyl-methyl)-benzamide cal'd 402 (MH+), exp402 (MH+)

Oxadiazoles

[0342]

[0343]4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoate. To a solution of difluoro[4-(methoxycarbonyl)phenyl]acetic acid (0.12 g, 0.52 mmol) and 2-methoxybenzohydrazide (0.13 g, 0.78 mmol) in methylene chloride (2 mL) was added 3-{[(ethylimino)methylene]amino}-N,N-dimethylpropane-1-aminium chloride (0.2 g, 1.04 mmol). The resulting solution was stirred at ambient temperature for 16 hours. The reaction was purified by flash chromatography (0-4% methanol/methylene chloride) to give methyl 4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoate as a white solid. ESIMS calcd 379.1 (M++H), found 379.1 (M++H).

[0344]4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoic acid. To a solution of methyl 4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoate (74 mg, 0.2 mmol) in THF (3 mL) was added sodium hydroxide (0.58 mmol, 0.58 mL of a 1M solution) and the solution was stirred at ambient temperature for 16 hours. The reaction was partitioned between 1M HCl and ethyl acetate. The organics were dried over sodium sulfate, filtered and evaporated to give 4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoic acid as a white solid. ESIMS calcd 365.0 (M++H), found 365.0 (M++H).

[0345]tert-butyl {3-[(4{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoyl)amino]-1-phenyl-1H-pyrazol-4 yl}carbamate. To a solution of 4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoic acid (65 mg, 0.18 mmol), tert-butyl (3-amino-1-phenyl-1H-pyrazol-4-yl)carbamate (74 mg, 0.27 mmol) and N,N-diisopropylethylamine (46 mg, 0.36 mmol) in methylene chloride (1.5 mL) was added (1H-1,2,3-benzotriazol-1-yloxy)(triisopropyl)phosphonium hexafluorophosphate (0.16 g, 0.36 mmol) and the reaction was sealed and heated to 60° C. for 16 hours. The reaction was purified by flash chromatography (0-4% methanol/methylene chloride) to give crude tert-butyl {3-[(4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoyl)amino]-phenyl-1H-pyrazol-4-yl}carbamate. ESIMS calcd 621.2 (M++H), found 621.2 (M++H).

[0346]tert-butyl {3-[(4-{difluoro [5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]methyl}benzoyl)amino]-1-phenyl-1H-pyrazol-4-yl}carbamate. To a solution of tert-butyl {3-[(4-{1,1-difluoro-2-[2-(2-methoxybenzoyl)hydrazino]-2-oxoethyl}benzoyl)amino]-1-phenyl-1H-pyrazol-4-yl}carbamate (0.05 g, 0.08 mmol) in THF (2 mL) was added Burgess reagent (38 mg, 0.16 mmol) and the reaction was sealed and heated to 100° C. for 10 minutes under microwave irradiation. The reaction was evaporated to dryness and purified by reverse phase chromatography (5/95 acetonitrile/water to 95/5 acetonitrile/water) to give tert-butyl {3-[(4-{difluoro[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]methyl}benzoyl)amino]-1-phenyl-1H-pyrazol-4-yl}carbamate as a white solid. ESIMS calcd 603.2 (M++H), found 603.2 (M++H).

[0347]N-(4-amino-1-phenyl-1H-pyrazol-3-yl)-4-{difluoro[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]methyl}benzamide. To a solution of tert-butyl {3-[(4-{difluoro[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]methyl}benzoyl)amino]-1-phenyl-1H-pyrazol-4-yl}carbamate (10 mg) in methylene chloride (2 mL) was added trifluoroacetic acid (1 mL) and the solution was stirred at ambient temperature for 16 hours. The reaction was evaporated to dryness to give N-(4-amino-1-phenyl-1H-pyrazol-3-yl)-4-{difluoro[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]methyl}benzamide as a yellowish solid. 1H NMR (CD3OD) δ 8.45 (s, 1H), 8.23 (d, J=8.21 Hz, 2H), 7.93 (m, 3H), 7.79 (d, J=7.62 Hz, 2H), 7.64 (m, 1H), 7.52 (t, J=7.62 Hz, 2H), 7.37 (t, J=7.62 Hz, 1H), 7.25 (d, J=8.5 Hz, 1H), 7.14 (t, J=7.62 Hz, 1H), 3.96 (s, 3H); ESIMS calcd 503.2 (++H), found 503.1 (M++H).

[0348]α-aminoaryl analogs from the carboxylic acids were prepared in procedures similar to those described for the preparations of the above examples. All compounds were prepared as the TFA salt form.

13 Cpd # Ra Name MS 13-1 2-OMe N-(4-Amino-1-phenyl-1H- cal'd 503.1 pyrazol-3-yl)-4-{difluoro-[5-(2- (MH+), exp 503.1 methoxy-phenyl)- (MH+) [1,3,4]oxadiazol-2-yl]-methyl}- benzamide

Procedure for B3. Compounds from (fluoro-alkoxycarbonyl-alkyl)-benzoic acids.

[0349]4-Carboxyoxalyl-benzoic acid methyl ester. To solution of 4-acetyl-benzoic acid methyl ester (15 g, 84.0 mmol) in pyridine (40 mL) at 90° C. was added selenium dioxide (16.4 g, 147 mmol) portionwise over 2 h. After 18 h, cooled to RT and filtered via Celite. The solvent was removed in vacuo and the residue was diluted with EtOAc, washed with 0.5N HCl, dried (MgSO4) and the solvent was evaporated under reduced pressure. The material was used without further purification. 1H NMR (DMSO-d6) δ 8.01 (d, J=8.2 Hz, 2H), 7.90 (d, J=8.2 Hz, 2H), 3.85 (s, 3H). cal'd 209 (MH+), exp 209 (MH+).

[0350]4-Methoxyoxalyl-benzoic acid methyl ester. To a solution 4-carboxyoxalyl-benzoic acid methyl ester (5.0 g, 24.0 mmol) and triethylamine (3.35 mL, 24.0 mmol) in CH2Cl2 (50 ml) at RT was added methyl chloroformate (1.86 mL, 24.0 mmol). After 30 min, the reaction mixture was diluted with CH2Cl2 (100 ml) and washed with H2O. The combined organic extracts were washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Biotage 25M), eluting with EtOAc/hexane to give a colorless solid. 1H NMR (CDCl3) δ 8.16 (d, J=8.2 Hz, 2H), 8.09 (d, J=8.2 Hz, 2H), 3.99 (s, 3H), 3.96 (s, 3H). cal'd 223 (MH+), exp 223(MH+).

[0351]4-(1-Hydroxy-1-methoxycarbonyl-ethyl)-benzoic acid methyl ester. To a slurry of 4-methoxyoxalyl-benzoic acid methyl ester (0.913 g, 4.11 mmol) in THF (20 ml) at −78° C. was added methyl magnesium chloride (2.74 mL, 8.22 mmol). During the addition the slurry became homogeneous. Let warm to RT overnight. The reaction was diluted with EtOAc and H2O— The combined organic fractions were dried (MgSO4), filtered and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel Biotage 25M, eluting with EtOAc/hexane to give as a colorless oil.

[0352]1H NMR (CDCl3) δ 8.01 (d, J=8.2 Hz, 2H), 7.63 (d, J=8.2 Hz, 2H), 3.90 (s, 3H), 3.78 (s, 3H), 1.78 (s, 3H). cal'd 239 (MH+), exp 239 (MH+).

[0353]4-(1-Fluoro-1-methoxycarbonyl-ethyl)-benzoic acid methyl ester. To a solution of 4-(1-hydroxy-1-methoxycarbonyl-ethyl)-benzoic acid methyl ester (365 mg, 1.53 mmol) in CH2Cl2 (5 ml) at 0° C. was added DAST (0.25 ml, 1.89 mmol). After 1.5 h, let warm to RT. The reaction was quenched with MeOH, diluted with CH2Cl2, washed with H2O. The organic layer was dried (MgSO4) filtered and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel Biotage 25M, eluting with EtOAc/hexane to give as a colorless oil. 1H NMR (CDCl3) δ 8.04 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H), 3.92 (s, 3H), 3.76 (s, 3H), 1.94 (d, J=22.3 Hz, 3H). cal'd 241 (MH+), exp 241 (MH+).

[0354]4-(1-Carboxy-1-fluoroethyl)-benzoic acid methyl ester. To a solution of 4-(1-fluoro-1-methoxycarbonyl-ethyl)-benzoic acid methyl ester (288 mg, 1.20 mmol) in THF/MeOH (2/2 mL) was added lithium hydroxide (1.26 mmol, 1.26 mL of a 1M solution) dropwise at ambient temperature. The resulting solution was stirred for 3 hours. The reaction was diluted with H2O and washed with EtOAc. The aqueous fractions were acidified with 1 N HCl and extracted with EtOAc. The organics were dried over sodium sulfate, filtered and evaporated to a white solid, which was used without further purification.

[0355]1H NMR (CDCl3) δ 8.06 (d, J=8.2 Hz, 2H), 7.61 (d, J=8.2 Hz, 2H), 3.92 (s, 3H), 1.98 (d, J=22.3 Hz, 3H). cal'd 227(MH+), exp 227(MH+).

[0356]4-(1-Fluoro-1-methylcarbamoyl-ethyl)-benzoic acid methyl ester. To a solution of 4-(1-carboxy-1-fluoro-ethyl)-benzoic acid methyl ester (200 mg, 0.884 mmol), HOBT (179 mg, 1.326 mmol), methylamine hydrochloride (179 mg, 2.65 mmol) and Et3N (0.431 mL, 3.09 mmol) in CH2Cl2 (5 mL) was added EDC (254 mg, 1-326 mmol). The mixture was stirred overnight. The solvent was removed. The residue was dissolved in EtOAc and washed with H2O, 0.5 N HCl, sat. NaHCO3, dried (MgSO4), filtered and the solvent was evaporated under reduced pressure. The residue was used without further purification. 1H NMR (CDCl3) δ 8.02 (d, J=8.2 Hz, 2H), 7.62 (d, J=8.2 Hz, 2H), 6.48 (br s, 1H), 3.90 (s, 3H), 2.83 (d, J=5.3 Hz, 3H), 1.98 (d, J=23.5 Hz, 3H). cal'd 240 (MH+), exp 240 (MH+).

[0357]α-aminoaryl analogs were prepared in procedures similar to those described for the preparation of the above 6-[(4-chloro-benzylcarbamoyl)-fluoro-methyl]-benzo[b]thiophene-2-carboxylic acid (4-amino-biphenyl-3-yl)amide. All compounds were prepared as the free base (parent) form.

14 Cpd # Ra Rb Name MS 14-1 NHMe Me N-(2-Amino-5-thiophen-2-yl- cal'd 398 (MH+), phenyl)-4-(1-fluoro-1- exp 398 (MH+) methylcarbamoyl-ethyl)-benzamide

Example 2

HDAC Inhibition by Novel Compounds

HDAC1-Flag Assay:

[0358]Novel compounds were tested for their ability to inhibit histone deacetylase, subtype 1 (HDAC1) using an in vitro deacetylation assay. The enzyme source for this assay was an epitope-tagged human HDAC1 complex immuno-purified from stably expressing mammalian cells. The substrate consisted of a commercial product containing an acetylated lysine side chain (BIOMOL Research Laboratories, Inc., Plymouth Meeting, Pa.). Upon deacetylation of the substrate by incubation with the purified HDAC1 complex, a fluorophore is produced that is directly proportional to the level of deacetylation. Using a substrate concentration at the Km for the enzyme preparation, the deacetylation assay was performed in the presence of increasing concentrations of novel compounds to semi-quantitatively determine the concentration of compound required for 50% inhibition (IC50) of the deacetylation reaction.

Example 3

HDAC Inhibition in Cell Lines

ATP Assay

[0359]The novel compounds of the present invention were tested for their ability to inhibit proliferation of the human cervical cancer (HeLa) and colon carcinoma (HCT 116) cells.

[0360]In this assay, also referred to as the Vialight Assay, cellular ATP levels are measured as a means of quantifying cellular proliferation. This assay makes use of a bioluminescent method from Cambrex (ViaLight PLUS, cat. #LT07-121). In the presence of ATP, luciferase converts luciferin to oxyluciferin and light. The amount of light produced (emission at 565 nM) is measured and correlates with a relative amount of proliferation. Human cervical cancer (HeLa) or colon carcinoma (HCT 116) cells were incubated with vehicle or increasing concentrations of compound for 48 hours. Cell proliferation was quantified by adding the cell lysis reagent (provided in the Vialight assay kit) directly to culture wells, followed by addition of the ATP-monitoring reagent (containing luciferase/luciferin). The amount of light produced is then measured (emission at 565 nM). The quantity of light produced, as measured by 565 nM absorbance, is directly proportional to the number of living cells in culture.

[0361]While this invention has been particularly shown and described with references to embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the meaning of the invention described. Rather, the scope of the invention is defined by the claims that follow.